Along with chemotherapy and surgery, radiation therapy (RT) is a leading treatment option for cancer. It is an element in the treatment plan of over 50 million cancer survivors worldwide. Even with advances in conformal image guided therapies, RT is still limited by damage to normal tissues. Both short and long term side effects have been associated with RT, including anemia, gastrointestinal distress, skin irritation, tissue fibrosis, and even secondary cancer. These compromise the quality of life for many patients during RT. RT kills cells through free radical generation that overwhelms cells' radical scavenging protective mechanisms, leading to DNA damage. As a preventive measure, antioxidant drugs have been discovered that can supplement cells' radical scavenging and reduce RT side effects. These drugs have their own side effects and whether they also protect tumor tissue is unclear, leading to controversy over their use. Cerium oxide nanoparticles (CONPs), made from an oxide of the rare-earth element cerium, may be a novel potential therapeutic for their application as protectors of normal tissue from RT. CONPs have been used as industrial catalysts, are well known for their unique redox chemistry, and have recently been investigated for their biomedical applications. CONPs have demonstrated protection of cells from free radicals produced by inflammatory, oxidative, and radiation damage, with minimal dose related toxicity. It has been shown that CONPs do not protect cancer cells from radiation damage, though the mechanisms of this loss of radio-protection are poorly understood. This project attempts to significantly increase understanding of these mechanisms by attempting to discover the effects of the tumor microenvironment (TME), specifically low pH and hypoxia, on CONPs' radio-protective properties. pH and hypoxia are the focus of this study because CONPs lose their redox properties at lower pHs, and the hypoxic conditions of the TME are critical to radical production and tumor radio-resistance and may also affect how CONPs react in tumor tissue. The effects of CONPs have been studied in vivo, but there is a lack of mechanistic studies under clinically simulating treatment conditions. This proposal will utilize multi-modality molecular imaging and conformal image guided RT to better understand the underlying mechanisms of CONPs' radio- protection in a spontaneously induced colon cancer mouse model. Molecular imaging will consist of SPECT- /PET-CT imaging of radiolabeled CONPs' (rCONPs) to measure uptake and pharmacokinetics, while MRI and tomographic photoacoustic imaging (TPA) will be used to determine in vivo pH and oxygen gradients. [18F]FDG PET imaging will be used to monitor tumor growth and tumor/normal tissue response to RT. Comparing the normal/tumor tissue response to the rCONPs' uptake and the pH/O2 conditions in vivo, with in vitro cross validation, will reveal how they are interrelated. The methods of this project are comparable to those available in the clinic and information from these studies may help accelerate CONP translation into RT clinical trials.